In the yeast Saccharomyces cerevisiae the expression of the galactose gene cluster (GAL 7, 10, 1) is highly inducible (100-500 fold). Genetic and biochemical studies have shown that expression of these genes (which code for the Leloir pathway enzymes) is under both positive (GAL 4) and negative (GAL 80) control by protein regulatory molecules. Available evidence suggests that GAL gene expression is regulated at the level of transcription. This system will be developed as a model for understanding, in biochemical detail, the molecular mechanisms controlling regulated eucaryotic transcription. This goal will be attained by first developing a soluble, yeast in vitro transcription system to assay for factors involved in directing specific transcription and then reconstructing these events utilizing purified or partially purified components. In order to carry out in vitro studies GAL DNA will be cloned and the transcriptional unit organization of these genes will be carefully mapped in order to define the GAL primary transcription products. The 5'-termini of the primary transcript(s) and promotor containing DNA fragments will be sequenced to define the transcription events which must be reproduced in vitro. Discrete transcripts synthesized in cell-free systems will be characterized in detail to document specific in vitro transcription initiation. The transcription system will then be fractionated to identify and purify accessory transcription components. The mechanism of action of these factors will be determined utilizing the purified components. These studies will serve to identify and characterize in molecular terms the components, and their mechanism of action, which are both necessary and sufficient for regulated eucaryotic transcription. This information will thus give us deeper insight into the mechanisms controlling eucaryotic gene expression and should have general significance for a large number of genetic systems.